This project is directed at understanding the mechanism of action of different ligands on branch DNA molecules. Small molecule ligands and proteins are of interest in this research. Sites of branching to form three- and four-arm DNA structures are important biological entities possessing unique physical properties that can be exploited to uncover new reagents for detecting, inhibiting or altering the yield of products in recombinational events. In the previous grant period we have established that a number of ligands, including many classical intercalators, some external binding drugs as well as metal complexes, interact tightly at the branch site in both three- and four-arm junctions. Blocking or altering the outcome of a recombination pathway by stabilizing normally transient intermediates is thus feasible and merits investigation. One major thrust of research in the next period concerns defining the structure of complexed and uncomplexed junctions to as high a resolution as possible. Details of the mechanism of binding and inhibition of resolution will be determined. Specific projects are the following: (1) Investigate the effects of tight binding ligands on the function of branched DNA molecules. (2) Determine the structure of three- and four-arm branched DNA's in the presence and absence of ligands including propidium, tetrapyridal porphyrins, and the thiacarbacyanin dye, Stains-All. Gel mobility assays will be used to define the geometry of junctions with and without ligands. Higher resolution structures for both the junctions and their ligand complexes will be obtained by NMR, with and without specific labeling of the junctions. (3) Determine complete thermodynamic profiles, kinetics and activation parameters for ligand interactions with three- and four-arm branched DNA, by means of an electrophoretic competition assay we have worked out and titration microcalorimetry. In the case of four-arm junctions, the effect of ligands on branch migration will be determined. In addition, if time and resources permit we will determine how ribose substitution at the branch affects the overall geometry and substrate properties of junctions; the mode of interaction of ligands with knots and ligated closed complexes of junctions relative to that in free junctions; and the potential of reactive ligands to cleave at the site of branching and thus resolve branched molecules.